Simvastatin and lovastatin are well known anti-hyperlipidemic or anti-hypercholesterolemic drugs and cholesterol lowering agent. Simvastatin is metabolized to at least four primary metabolites, namely 6′β-OH simvastatin, 6′-exomethylene simvastatin, 6′-hydroxymethyl metabolite, and 3′-OH simvastatin. Although CYP3A4 is the main enzyme involved in the primary metabolism of simvastatin, CYPs 2C8 (Tornio et al., 2005), 2C9 (Transon et al., 1996), and 2D6 (Transon et al., 1996) are also involved in the formation of simvastatin metabolites.

The extensive oxidative metabolism of lovastatin in the human liver is primarily mediated by CYP3A enzymes, particularly CYP3A4, to generate three known metabolites, namely 6′β-OH lovastatin, 3″-OH lovastatin, and 6′-exomethylene metabolites (Garcia et al., 2003; Caron et al., 2007).

After oral ingestion, simvastatin and lovastatin, which are inactive lactones, are hydrolyzed to the corresponding mvastatin and lova (Vickers et al., 1990a). This is a principal metabolite and an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. This enzyme catalyzes the conversion of HMG-CoA to mevalonate, which is an early rate limiting step in the biosynthesis of cholesterol. In addition to the P450-mediated oxidation and β-oxidation processes, glucuronidation constitutes a common metabolic pathway for statins (Prueksaritanont et al., 2002). It was found that the metabolites resulting from microsomal oxidation of simvastatin (Vickers et al., 1990) and lovastatin (Vyas et al., 1990) by P450 enzymes are effective inhibitors of the HMG-CoA) reductase. Therefore, it was suggested that the metabolites may contribute to the cholesterol lowering effect of simvastatin and lovastatin. However, systematic studies of safety, efficacy, and toxicity of the metabolites have not yet been carried out. The major metabolites including 6′β-OH statins, have not been prepared by chemical synthesis previously.
Cytochrome P450 enzymes (P450s or CYPs) constitute a large family of enzymes that are remarkably diverse oxygenation catalysts found throughout nature, from archaea, bacteria, fungi, plants, animals and humans http:drnelson<dot>utmen<dot>edu/CytochromeP450<dot>html). Due to their catalytic diversity and broad substrate range, P450s are attractive as biocatalysts in the production of fine chemicals, including pharmaceuticals (Guengerich 2002; Urlacher et al., 2006; Yun et al., 2007; Lamb et al., 2007). In spite of the potential use of mammalian P450s in various biotechnology fields, they are not suitable as biocatalysts mainly because of their low stability and low catalytic activity.
If prodrugs are converted to biologically ‘active metabolites’ by human liver P450s during the drug development process (Johnson et al., 2004), the pure metabolites are required to understand the drug's efficacy, toxic effect, and pharmacokinetics. When the pure metabolites are difficult to synthesize by chemical methods, using the P450s is a useful alternative to generate the metabolites of drugs or drug candidates. Metabolite preparation has been demonstrated using human liver P450s expressed in Escherichia coli (Yun et al., 2006) and in insect cells (Rushmore et al., 2000; Vail et al., 2005), but these systems are still costly and have low productivities due to limited stabilities and slow reaction rates (Guengerich et al., 1996). It was shown that engineering bacterial P450 BM3 could produce human drug metabolites (Yun et al., 2007 and references therein; Kim 2009; Kim 2010; Park 2010). Recently, the Food and Drug Administration (FDA) modified its standards for evaluating drug toxicity, particularly with regard to the toxicity of drug metabolites. In February 2008, the FDA issued the Guidance for Industry: Safety Testing of Drug Metabolites (Food and Drug Administration, Guidance for Industry: Safety Testing of Drug Metabolites; www<dot>fda<dot>gov/downloads/Drugs/Guidance ComplianceRegulatoryInformation/Guidances/ucm079266<dot>pdf). According to this guide, any human drug metabolites “ . . . formed at greater than 10 percent of parent drug systemic exposure at steady state should be subject to separate safety testing, that is, by synthesis and administration to test animals (Guengerich, 2009 and references therein). The issue of human metabolites in safety testing (MIST) has presented a challenge at the early stages of drug development for the pharmaceutical industry. Some metabolites of concern can be prepared by chemical methods, but the others may not be easily prepared by the chemical methods. In the later cases, human liver microsomes, heterologously expressed human enzymes in bacteria, and purified human enzymes might be good candidates for biocatalysts to prepare human drug metabolites. However, they have several weaknesses such as low catalytic activity and low stability for industrial use to prepare the metabolites.
All the cited references are incorporated herein by reference in their entireties. The information disclosed herein is intended to assist understanding of the technical background of the present invention, and cannot be prior art.